Migration of epithelial cells occurs during tissue morphogenesis, wound healing and metastasis of epithelial tumors. The onset of migration requires integration of a complex set of signals derived from interactions with the extracellular matrix, neighboring cells and with hormonal or other soluble factors. Members of the Rho family of GTPases have been shown to be important integratrs of these signals, translating them into changes in the actin cytoskeleton that are required for cell motility. We and others have recently shown that a second class of small GTPases, the ADP-ribosylation factors (ARFs) can also regulate assembly of the actin cytoskeleton and appear to do so coordinately with Rho family members. Using MOCK cells as a model epithelium, we found that activation of ARF6 (by expression of the nucleotide exchange factor ARNO) is sufficient to induce a migratory phenotype similar to that of cells treated with scatter factor/HG F. Conversely, migration induced by either wounding of epithelial monolayers or by HGF treatment is inhibited by a dominant negative ARF6 construct. Migration requires two downstream events initiated by ARF6, activation of the Rho GTPase Raci and activation of the ARF effector, phospholipase D. Importantly, Raci activation is not dependent on PLD, and PLD activation is not dependent on Raci, suggesting that each represents a distinct signaling pathway downstream of ARF6. These data suggest that ARF6 plays a central role in the regulation of epithelial cell motility. The overall goal of this proposal is to determine the molecular mechanisms through which ARF6 regulates motility. Three specific aims are proposed: 1) We will define the role of PLD in cell migration by identifying the specific PLD isoform(s) and the specific metabolic products involved; 2) We will determine the molecular mechanism by which ARF6 activates Raci, as well as the regulation by ARF6 of a muttiprotein complex consisting of an ARF GAP (GIT), the Rac nucleotide exchange factor PIX, the Rac effector kinase PAK and the focal adhesion component paxillin; 3) We will define the role of GIT tyrosine phosphorylation in the transmission of ARF6-derived signals through the GIT/PIX/PAK/paxillin complex.